Takara SMART-Seq mRNA LP (with

SMART-Seq mRNA LP (with UMIs) generates oligo(dT)-primed, full-length mRNA-seq libraries with UMIs, providing greater accuracy for quantitative gene expression analysis across samples, while controlling for PCR errors and amplification biases. The chemistry is optimized for use on ultra-low amounts of total RNA (10 pg–100 ng, RIN≥8) or for direct use on multiple intact cells (<1,000 cells). Up to 384 multiplexed, Illumina-ready sequencing libraries can be obtained using theUnique Dual Index kits (Cat. Nos. 634752–56). This kit offers an end-to-end solution including cDNA synthesis, library preparation, and data analysis with our free Cogent NGS bioinformatics tools.

The SMART-Seq chemistry efficiently captures the 5 end of transcripts. UMIs are added during the first strand synthesis. High-quality, Illumina-ready sequencing libraries can be prepared from single cells and ultra-low amounts of RNA.

The original SMART-Seq mRNA LP kit has an update. The addition of UMIs provides higher accuracy RNA counting. Detect rare transcripts and identify rare cell types with higher confidence.

The SMART-Seq mRNA LP (with UMIs) kit workflow goes from cDNA synthesis to library prep. Sequence on an Illumina instrument and use the resulting FASTQ files as input for our free analysis software. Cogent NGS Analysis Pipeline is used for mapping and reporting. Cogent NGS Discovery Software is used for interactive data visualization.

SMART-Seq mRNA LP (with UMIs) and SMART-Seq mRNA LP were each used to create sequencing libraries from K562 RNA (10 pg) or universal human reference (UHR) RNA (100 pg, 1 ng, 10 ng, and 100 ng). Gene counts and RNA subtype representation (read distribution) were similar across the two methods.

cDNA and sequencing libraries were generated from 10 pg universal human reference (UHR) RNA using the SMART-Seq mRNA LP (with UMIs) (SSmRNA +UMIs) or the SMART-Seq2 (SS2) method. The latter were prepared using the Nextera XT DNA Library Prep Kit (Illumina). After pooling, sequencing, and downsampling to 1.2 million reads, SSmRNA +UMIs resulted in a higher gene count with similar read distribution.

Absolute read counts were calculated, and regression analysis was performed for each replicate pair using our free bioinformatic software, Cogent AP. The R2values were consistently higher for SMART-Seq mRNA LP (with UMIs) than for Smart-seq2.

10 pg of input RNA or RNA from a single cell was spiked with ERCC control RNA. Libraries were prepared using the SSmRNA +UMIs method. After sequencing, our free bioinformatic software, CogentAP, was used to plot TPM or CPM against the absolute number of input molecules from the ERCC spike-in. Subsequent regression analysis showed a significant increase in the R2value upon UMI collapse.

Single peripheral blood mononuclear cells (PBMCs) were sorted into plates. Sequencing libraries were produced using either SMART-Seq mRNA LP or SMART-Seq mRNA LP (with UMIs). Libraries were then pooled, sequenced, and downsampled to 900,000 reads for analysis. The comparability of gene counts read distributions illustrates that data quality is not compromised by including UMIs.

Required Products

The Unique Dual Index (UDI) Kit (1–96) contains 96 pairs of unique dual-indexed PCR primers that can be used to generate up to 96 Illumina-compatible sequencing libraries and are recommended with several of Takara Bio’s RNA-seq and DNA-seq kits.

The Unique Dual Index (UDI) Kit (97–192) contains 96 pairs of unique dual-indexed PCR primers that can be used to generate up to 96 Illumina-compatible sequencing libraries and are recommended with several of Takara Bio’s RNA-seq and DNA-seq kits.

The Unique Dual Index (UDI) Kit (193–288) contains 96 pairs of unique dual-indexed PCR primers that can be used to generate up to 96 Illumina-compatible sequencing libraries and are recommended with several of Takara Bio’s RNA-seq and DNA-seq kits.

The Unique Dual Index (UDI) Kit (289–384) contains 96 pairs of unique dual-indexed PCR primers that can be used to generate up to 96 Illumina-compatible sequencing libraries and are recommended with several of Takara Bio’s RNA-seq and DNA-seq kits.

The Unique Dual Index (UDI) Kit (1–24) contains 24 pairs of unique dual-indexed PCR primers, which are a subset of the Unique Dual Index Kit (1–96), Cat. No. 634752. They can be used to generate up to 24 Illumina-compatible sequencing libraries and are recommended with several of Takara Bio’s RNA-seq and DNA-seq kits.